The present invention is generally related to the field of cytological and molecular assays and specifically to the area of assays for the assessment of disease using a sensitive assay for diagnosis and prognosis of HPV-induced carcinoma.
The detection and diagnosis of disease is of obvious importance for the treatment of disease. Numerous characteristics of diseases have been identified and many are used for the diagnosis of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can include the expression of viral genes in infected cells, changes in the expression patterns of genes in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the assessment of such cell states.
An aspect of the present invention relates to human papilloma virus (HPV), which induces benign epithelial proliferations of the skin and mucosa in humans and is associated with anogenital neoplasias and carcinomas. The intact DNA of HPV is supercoiled and thus resembles an endless loop of twisted telephone handset cord. Inside this shell, the viral DNA is packaged in and around proteins from the cell nucleus, histones, and associated peptides, into a structure that resembles cellular chromatin. (Turek, (1994)). Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types. HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer. All human and animal papillomaviruses appear to share a similar genetic organization, although there are differences in the functions of individual viral genes and in their regulation. The most common genital HPV type associated with cervical carcinoma, HPV 16, has been studied most extensively.
All large open reading frames (ORFs) in HPV are on one DNA strand. Papillomaviral mRNAs appear to be transcribed solely from a single strand in infected cells. The viral genome can be divided into three regions, the upstream regulatory region (URR), or long control region (LCR), containing control sequences for HPV replication and gene expression, the viral early gene region, encoding, among others, the E2, E6 and E7 genes, and the late region, encoding the L1 and L2 genes. (Turek, (1994)).
HPV gene expression in high-grade premalignant disease or cancer appears restricted to the early genes, possibly due to cellular differentiation arrest induced by the viral E6 and E7 genes. In comparison to active HPV infection, E6 and E7 gene control in cancer is deranged by mutations in the viral URR and, in integrated viral fragments, by the disruption of the viral E2 gene, stabilization of E6 and E7 mRNAs, and influences at the cellular integration site.
Because the E2 gene is disrupted or inactivated in integrated HPV fragments in invasive cervical carcinomas (Cullen, (1991); Dxc3xcrst, (1985); Matsukura, (1989); Schneider-Gxc3xa4dicke, (1986); Schwarz, (1985); Wilczynski, (1988)), it has been predicted that loss of E2 bestows a selective growth advantage to the infected cell because of uncontrolled E6 and E7 expression (Schneider-Gxc3xa4dicke. (1986); Schwarz, (1985)). Indeed, cervical cells containing replicating HPV genomes rapidly segregate and are outgrown in culture by cells that contain integrated viral genomes (Jeon (1995)), but the underlying mechanism(s) have remained unclear until recently. The full-length HPV 16 E2 gene products are strong transcriptional activators comparable to HPV 1 E2 at some viral as well as at simple, synthetic promoters (Demeret (1994); Ushikai (1994)).
Genes E6 and E7 are considered to have oncogenic activity. The encoded proteins interact with and disturb the physiologic functions of cellular proteins that are involved in cell cycle control. The E6/E7 proteins of HPV 16, 18 or related types are most efficient in this regard. Some of these activities lead to genetic instability of the persistently infected human cell. This enhances the probability of mutations in cellular proto-oncogenes and tumor suppressor genes and thus contributes to tumor progression. Mutations in cellular genes devoted to the intracellular surveillance of HPV infections, integration of viral DNA, and deletions or mutations of viral transcription control sequences lead to a significantly increased expression of the E6/E7 genes, which is a consistent characteristic of high-grade intraepithelial neoplasia and cancers. The genetic instability caused by viral oncoproteins and the autocatalytic increase in oncoprotein expression caused by mutations in the viral and cellular genome identify the virus as a major driving force of progression to carcinoma.
Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is used for identifying patients with premalignant lesions who are at risk of progression to malignancy. Although visible anogenital lesions are present in some persons infected with human papillomavirus, the majority of individuals with HPV genital tract infection do not have clinically apparent disease, but analysis of cytomorphological traits present in cervical smears can be used to detect HPV infection. Conventional viral detection assays, including serologic assays and growth in cell culture, are not commercially available and/or are not suitable for the diagnosis and tracking of HPV infection. Papanicolaou tests are a valuable screening tool, but they miss a large proportion of HPV-infected persons.
Thus, it is an object of the present invention to provide a method for assessing the stage of HPV-based disease.
It is another object of the present invention to provide an assay that can be combined with other assays to improve the accuracy and reliability of prognostic and diagnostic assessments of HPV-based disease.
It is a further object of the present invention to provide a method for assessing the risk that a patient infected with HPV will develop HPV-based disease.
It is another object of the present invention to provide a method for stratifying patients who are currently HPV-infected but without detectable HPV-based disease into those at risk for progression to disease and those not at risk for progression to disease.
It is also an object of the present invention to provide a method for identifying treatment regimes for patients having HPV-based disease.
Yet another object of the present invention to provide a method for monitoring the effectiveness of treatment of HPV-based disease.
A further object of the present invention to provide kits for assessing the stage of HPV-based disease.
Another object of the present invention to provide computer-based operation, analysis, and data management of assay data to assess the stage of HPV-based disease.
One embodiment of the present invention involves measuring the levels of expression of genes involved in a cell state, and comparing their expression to each other or to reference genes in a specific ratio, as an indication of the state of a disease in the cell sample. The present invention can be used to assess the stage or risk of a disease as indicated by the state of the cells. It can also be used to guide or assess the effectiveness of a therapy for a disease by identifying appropriate therapy based on the indicated cell state or by indicating any change in the state of cells subjected to the therapy.
In one form of the present invention, the stage and prognosis of a human papillomavirus (HPV) infection or HPV-based disease is assessed. This embodiment involves the measurement of the level of expression of two or more HPV genes. Genes for this purpose are the HPV E6, E7, L1, and E2 genes, although other HPV genes such as E1, E4, E5, and L2 can also be used. It has been discovered that the level of expression of these genes, the ratio of expression of these genes to each other or to reference genes, or both, are indicative of the stage of HPV-based disease.
Gene expression levels are used according to this invention to assess the progression of HPV infection from benign to malignant growth. HPV infection progresses from CIN I through CIN III and finally to malignant cancer. These stages can be identified by the ratios of HPV genes. In particular, the transition from CIN I to CIN II/III, i.e., a transition to pre-malignancy, can be predicted when the ratio of the present invention exceeds one. A ratio of greater than 3 in the present invention indicates a transition from pre-malignancy to malignant cancer. Thus, a ratio below 1 indicates a low-level of CIN in an HPV infected cell. A ratio between one and about three indicates a high grade CIN (i.e., CIN III) or pre-malignant condition. And a ratio of over about three is an indication of an HPV induced malignancy.